Final Exam Study GuideBelow, you will find a list of learning objectives to emphasize from the cumulative material that will be covered on the remaining portion of the final exam. They are listed in order as they were presented in lecture and are broken down into categories. You will find that some of these learning objectives bridge multiple sections and are redundant or related to each other. In addition, you will find that the material we are learning in this final section will apply to several of these earlier learning objectives as well, with the goal of helping you tie together many of the concepts we have discussed across the semester! Think about how you can continue to integrate what we have learned.Study suggestions for the final examPrepare the new material as you would for a midterm, reviewing lecture notes, slides, relevant book chapters and articles (from the reading assignments), and in-class activities (including the non-graded ones too!). The emphasis, as it has been in the past, will be on material covered in lecture.For reviewing the cumulative material from previous sections here are a few study ideas and suggestions to consider (many of which will overlap with what you have been doing already!): Review lecture slides and notes Use the textbook and articles as resources where you need them: consider re-reading sections where you need clarification for your notes/slides and sections that you found most challenging for you on previous exams Write out information relevant to the learning objectives to help you assess which learning objectives you need to spend more time reviewing Use the figures and graphs in the lecture slides and text book to practice explaining and describing what they show Go back and review any in-class activities Flash cards for terminology are a great way to review and practice recalling information if you find it challenging to remember details from previous sections Form a study group and quiz each other on relevant material Concept map and draw out and detail pathways Think about how you can make connections from one section to another, compare and contrast processes

Techniques List several uses of antibodies in scientific research Allows for precise visualization of selected proteins among the many thousands that each cell typically produces Individual antibody-producing B lymphocytes from an immunized mouse or rat, when fused with cells derived from a transformed B lymphocyte cell line, can give rise to hybrids that have both ability to make a particular antibody and the ability to multiply indefinitely in culture. A monoclonal antibody can be made against any protein in a biological sample. Once an antibody has been made, it can be used to localized the protein in cell and tissues, to follow its movement, and to purify the protein to study its structure and function. Compare and contrast methods of protein identification Use of protein tags for purification- using standard genetic engineering techniques, a short peptide tag can be added to a protein of interest. If the tag is itself an antigenic determinant, or epitope, it can be targeted by an appropriate commercially available antibody. The antibody, suitably labeled, can be used to determine the location of the protein in cells or to purify it by immunoprecipitation or affinity chromatography. In immunoprecipitation, antibodies directed against the epitope tag are added to a solution containing the tagged protein; the antibodies specifically cross-link the tagged protein molecules and precipitated them out of solution as antibody-protein complexes.

Separation by SDS-PAGE- Type of electrophoresis used to separate proteins by size. The protein mixture to be separated is first treated with powerful negatively charged detergent (SDS) and with a reducing agent (B mercaptoethanol), before being run through a polyacrylamide gel. The detergent and reducing agent unfold the proteins, free them from association with other molecules, and separate the polypeptide subunits. Western Blotting (Immunoblotting)- technique by which proteins are separated by electrophoresis and immobilized on a paper sheet and then analyzed, usually by means of a labeled antibody. Mass Spectrometry- Technique for identifying compounds on the basis of their precise mass-to-charge ratio. Powerful tool for identifying proteins and sequencing polypeptides.

Describe the major methods used to analyze gene expression and alter gene function Analyze gene expression RNA isolation and cDNA production- total mRNA is extracted from a particular tissue, and the enzyme reverse transcriptase produces DNA copies (cDNA) of the mRNA molecules. A short oligonucleotide complementary to the poly-A tail at the 3 end of the mRNA is first hybridized to the RNA to act as a primer for the reverse transcriptase, which then copies the RNA into a complementary DNA chain, thereby forming a DNA/RNA hybrid helix. Treating DNA/RNA hybrid with RNase H creates nicks and gaps in the RNA strand. The enzyme DNA polymerase then copies the remaining single-stranded cDNA into double-stranded cDNA. The fragment of the original mRNA is the primer for this synthesis reaction, as shown. Because the DNA polymerase used to synthesize the second DNA strand can synthesize through the bound RNA molecules, the RNA fragment that is base-paired to the 3 end of the first DNA strand usually acts as the primer for the final product of the second strand synthesis. This RNA is eventually degraded during subsequent cloning steps. As a result, the nucleotide sequences at the extreme 5 ends of the original mRNA molecules are often absent from cDNA libraries.

Polymerase Chain Reaction (PCR)- technique for amplifying specific regions of DNA by the use of sequence-specific primers and multiple cycles of DNA synthesis, each cycle being followed by a brief heat treatment to separate complementary strands. Conventional PCR-

Quantitative PCR- Red has higher gene expression. It amplifies sooner Microarray analysis- a large array of short DNA molecules (each of known sequence) bound to a glass microscope slide or other suitable support. Used to monitor expression of thousands of genes simultaneously: mRNA isolated from test cells is converted to cDNA, which in turn is hybridized to the microarray.

Alter gene function pg. 567

RNA molecules being used to inhibit gene expression (siRNA) Use knowledge of established methods to suggest how to test a research question analyzing regulation of gene expression or protein functionProtein Function Explain how proteins containing similar protein domains have diversified function In addition to gene duplication, domain shuffling has diversified proteins containing similar domains. Shuffling of blocks of protein domains has occurred during protein evolution. Processes such as recombination and splicing have lead to diversified function (s).

Describe the significance of binding specificity for antibody recognition of antigen The more compatible they are, the more non-covalent interactions will form which means that longer associations will occur. If binding is not specific, very short or no interaction will occur. Outline two ways that phosphate groups can be used to regulate protein function Can turn on/off protein function by covalent addition of a phosphate group to an amino acid side chain.

Can induce a conformational changes to more tightly regulate signaling pathway

Activation of Src kinase- Messing up this cascade would lead to cell proliferation uncontrollably. Membrane Transport and Intracellular Trafficking List the transport mechanisms by which molecules can pass through the cell membrane Track the pathway of a membrane protein to the cell surface and identify several points of regulation in the pathway Determine what guides vesicles to their target location Identify and describe how pathogens usurp the normal immune function in place to protect the host from these pathogens Cell Signaling Suggest how GPCRs may have evolved to regulate taste perception in cells on the tongue and provide protective responses in the airways Taste receptor signaling- from tongues to lungs (Kinnamon, Acta Physiol. 2012) Taste receptor signaling is not confined to the taste buds on the tongue, found so far in several other locations Airways Pancreas Brain Gastrointestinal tract Bitter receptors Evolved to help the organism avoid toxic (bitter) substances Detected in the lungs by used of GFP-reporter mice E.g. GFP under control of a-gust and T1R3 promoters Cell types in airways expressing Bitter receptors: Solitary chemosensory cells (SCCs), ciliated epithelial cells, and smooth muscle cells SCCs mostly restricted to vertebrates In the tongue, most buds express one set/kind of receptor, but in airways, the cells express both types

Nuclear export Describe the known mechanisms regulating nuclear export of mRNAs NPCs allow bidirectional transport Diffusion Water, sugar, ions, small molecules Facilitated Transport Larger molecules (e.g. mRNPs, rRNA, proteins) 4 NPC-mediated export pathways have been described CRM1-dependent; 4E;SE CRM1-dependent; ARE CRM: Ran GTP DEPENDENT CRM1-dependent; NXF3-mediated NXF1-dependent NXF1/NXT1: Ran GTP independent Most mRNAs are thought to use this pathway

Suggest a potential function for the newly described nuclear envelope budding model Export of mRNA by nuclear envelope budding Budding of inner membrane into lumen of the nuclear envelope, then vesicular fusion with the outer nuclear membrane Herpesvirus (HSV): nuclear egress Virons in this case use a similar process thought to be virus specific Perhaps HSV was able to mimic or take advantage of a process that already existed in the host cell. Proposed budding-mediated mRNA export PROCESS: Phosphorylation of nuclear lamins is suspected to be involved in this process (atyplical PKC) Identified by a study investigating development of Drosophila larval synapse formation Wnt-dependent neuromuscular junction synapse formation RNP complexes that function in neuromuscular junction formation export the nucleus through budding of the nuclear membrane DFrizzled (Wnt receptor) in muscle cells binds to ligand (Wingless, Wg) released from motor neuron, to form neuromuscular synapse during development Upon binding Wg, the receptor is internalized and the C-term cleavage product (DFz2C) is released, then imported to the nucleus RNP granules buds from nucleus and exocytose into the cytosol in the muscle cells via lamin C thru perinuclear space DFz2C foci formation is dependent on Lamin C Experimenters used PABP-GFP fusion protein to determine if RNA was present in the granules PABP-GFP is a Poly(A) binding protein aPKC plays in role in phosphorylation of lamin HINT from HSV LamC mutant cells do not have postsynaptic Par6 proteinCell Death Suggest how cells in the human body may utilize necroptosis to defend against intracellular pathogens Compare and contrast apoptotic and necroptotic cell death pathwaysCell Adhesion Describe the role of cell-cell adhesion during an immune response to mediate inflammation Selectins and integrins involved in tethering and rolling, the remainder of the pathway is complicated, but involves a variety of interactions between the lymphocytes and the endothelial cells (heterophilic interactions) Tethering and rolling occurs between leukocytes and endothelial cells until firm a point of firm adhesion is reached. After this, diapedesis occurs which is a process of cells attaching and going through 2 endothelial cells. Describe the basic events that occur during an inflammatory response to infection and how this leads to increased vascular permeability 1) Tissue Damage Occurs 2) Tissue damage causes release of vasoactive and chemotactic factors that trigger a local increase in blood flow and capillary permeability. 3) Permeable capillaries allow an influx of fluid (exudate-complement, antibody, C-reactive protein) and cells 4)Phagocytes migrate to site of inflammation (chemotaxis) 5) Phagocytes and antibacterial exudate destroy bacteria 6) Tissue Repair occurs General properties: The bodys defense mechanism in response to tissue damage and/or infection Initiators: infection, trauma (physical or chemical), and pathologic immune responses Purpose: identify the area affected and call into play mechanisms that lead to (a) elimination of the inflammatory stimulus and (b) healing Selectins and integrins important here White blood cells and endothelial cells interact with surface receptors (selectins) Once rolling and strongly adhered (integrins), diapedesis/extravasation occurs

Suggest how decreasing vascular permeability may be advantageous for combat severe flu infections Decreasing vascular permeability may be advantageous for combat severe flu infections because one wouldnt have the issue of dealing with leukocytes which have been shown to release cytokines, ROS, elastase, and amino acids which damage the endothelium barrier. Identify cell adhesion/junction components that may serve as promising drug targets for development of future therapeutics Slit2N: increases retention of VE-cadherin at the cell-cell endothelial junctions Doxycycline: increases expression of VE-cadherin Both of these have been tested for these functions in mouse models and shown improvement increased survival and/or decreased symptoms Can target ENaC- agonist which would lead to help clear fluid from lungs

Cell Cycle Describe at least three techniques that can be used to study cell division and/or cell cycle progression and suggest when these techniques may be useful Count cells to determines cell density Dead cell exclusion by Trypan Blue staining Trypan blue stains dead cells. Dying cells allow access of the outside to the inside. How would dying cells affect interpretation of your cell counts? How quickly cells divide (rate) and rate at which theyre dying influences count. This approach is not good because cells could replicate rapidly and then die instantly. BrdU Artificial thymidine analog incorporated into replicating DNA. Instead of thymidine, BrdU is added to interact with adenine. Short vs. long pulse Short pulse to figure out how many cells are dividing in short period of time. Long pulse to determine how many cells are dividing over a longer period of time Visualize with anti-BrdU antibody Can determine what % of cells are proliferating and timing of progression through cell cycle stages with flow cytometry CFSE (Carboxyfluorescein succinimidyl ester) Fluorescent dye that binds to proteins in the cell Can monitor the number of cell divisions of CFSE will go to a daughter cell and to the other. Each round of division you half the amount of CFSE DNA can be labeled with a dye too- allowing analysis of DNA content in cells by flow cytometry. Example: using propidium iodide

Are these cells proliferating? Yes. All cells in G1 phase peak have a relative amount of 1 DNA. Second peak shows # of cells whose DNA has been replicated already. Level of dye detected is equivalent to amount of DNA present. Cells in second peak are closer to getting duplicated.Would not be able to tell difference between cells in G1 and G0 Can follow cell cycle in vitro Following two proteins that function to regulate interphase in human fibroblasts by time-lapse live cell imaging: Cdt1 is labeled red: protein expressed during G1 and early S phase Geminin is labeled green: S/G2 expression So, cells are red during G1, yellow during S, and green in G2 -> then the cells enter M phase to divide. Yellow seen if there is overlapping Cdt1 and Geminin proteins are localized in the nucleus. Once accumulated, cells stopped dividing and were red (stuck in G1). Cells ran out of space.

Define the roles of cyclins, Cdks, and Cdk inhibitors and determine how alteration in expression or function of any of these regulators will influence cell cycle control Cyclins- protein that periodically rises and falls in concentration in step with the eukaryotic cell cycle. Cyclins activate crucial protein kinases(Cdks) and thereby help control progression form one stage of the cell cycle to the next. Cdks- Protein kinase that has to be complexed with cyclin protein in order to act. Different Cdk-cyclin complexes trigger different steps in the cell-division cycle by phosphorylating specific target proteins. Cdk inhibitors- protein that binds to and inhibits cyclin-Cdk complexes, primarily involved in the control of G1 and S phases. NEW MATERIAL

Ovarian Cancer Case Study Determine ways to test if a cancer marker serves a function in the development or maintenance of the cancer or if it is simply a byproduct Describe two factors associated with prognosis for ovarian cancer patients undergoing surgical removal of their tumor and chemotherapy Discuss the types of mutations and changes to the cell that must occur to develop an ovarian cancer, including the role of oncogenes, tumor suppressor genes, and EMT/MET transitions Suggest why ovarian cancer treatments are not successful in preventing cancer reoccurrences in some patients Compare and contrast several current treatments/therapies in use and in the pipeline

Hematopoiesis Explain how blood cells are derived from hematopoietic stem cells in the bone marrow Hematopoietic stem cells (HSC) become Multipotential Stem Cells (MSC) which then differentiates into Lymphoid Progenitor Cells (CLP-common lymphoid progenitor) OR Myeloid Progenitor Cell (MPC- common myeloid progenitor). CLP later differentiates into Natural Killer Cells (NK), T lymphocytes, B lymphocytes or dendritic cells. MPC goes on to differentiate into Granulocytes (Neutrophils, Basophils, Eosinophils), platelets, monocytes, mast cells, dendritic cells, or red blood cells (erythrocytes). Describe how bone marrow reconstitution was used to identify blood cell precursors Mice were injected with IVs that were constituted of common lymphoid progenitor (CLP) cells and HSC. This graph showed that more CLPs showed reconstitution of B and T cells faster in the spleen because theyre already on their way there. In contrast, HSC showed more reconstitution at a later time. The experiment tells us that CLP is not prolonging the production the way that HSC is. CLP is not going to be able to fully reconstitute the way the stem cell can in terms of the duration. HSC differentiate and renew. CLP cant renew. The second graph shows how T cells develop from observing CLP and HSC cells. In CLP, by 6 weeks, you have all mature T cells. Takes at least 3 weeks to detect progenitor cells in the thymus. CD4 cells develop faster. Once again, CLP are showed to differentiate faster. In end results, both will have pretty similar outcome (more CD4). The graph shows density of cells and percentages of each type. Bottom left quadrant = DN = double negative. Top right= DP= double positive. Other two are SP= single positive (for either CD8 or CD4). Top left is CD4, bottom right CD8. Identify two important features of hematopoietic stem cells that allow for life long production of new blood cells HSC must be able to self- renew and differentiate. Stromal cells in bone marrow support HSC maintenance: survival and self-renewal. Stromal cells will interact with HSC to keep them alive and in their undifferentiated state. The dependence of HSCs on stromal cell signals will limit the availability of HSC niches in the bone marrow.

Describe the role of signals from the extracellular environment and the role of transcription factors during the stepwise differentiation of the blood cells lineages HSC and its progenitors know whether to go down myeloid/lymphoid pathway depending on signals it receives from the environment (colony stimulating factors).

There are specific receptors that will bind to cytokines and then they take use of common/shared subunits to put together final signaling complex. If a progenitor cell has ability to give rise to multiple lineages, it may express a variety of receptors on its surface and then together with a common secondary binding partner depending on which ligand is present. The signal that results from activation is what tells the cell to do something.

The role of signals from extracellular environment is to cause a change in gene expression in order to produce cells that are needed at the moment. Transcriptional regulation of hematopoiesis Lineage fate decisions Developmental checkpoints ex: lineage and stage specific recombination of Ig (surface antibody on surface of B cell) and TCR genes in B and T cells, respectively Establishment of gene expression profiles Determines cell specific function. Transcriptional regulation is a process of how we get cell specific functions. Transcription factors determine lineage fate and drive cell specific characteristics and functions Suggest why hematopoietic progenitor cells can be the source of leukemias when regulatory pathways are disrupted Enhancing what a stem cell already does is going to be compatible with developing a cancer. Deregulated pathways leading to leukemia. Although leukemias are heterogeneous in terms of phenotypes, there are general mechanisms underlying leukemic transformation such as increased cell survival, increased proliferation capacity, increased self-renewal capacity, genomic instability, and prevention of differentiation. Examples of such deregulated mechanisms and/or signaling pathways that have been found in various types of leukemias are indicated.

Innate and Adaptive Immunity List the main cellular and molecular components of innate and adaptive immunity and the general functions of each component Innate, non-specific defenses Physical and/or chemical barriers, e.g., epithelia Cellular - phagocytic cells, natural killer (NK) cells (patrol for virally infected or cancer forming cells) Molecular - cytokines, chemokines, acute phase proteins, complement Acquired, adaptive, specific defenses Molecular antibodies (secreted by B cells) Cellular lymphocytes (naive, effector, and memory lymphocytes), help from phagocytic cells Innate and Acquired are NOT mutually exclusive Explain how the innate immune response functions to elicit an adaptive response when necessary Recognition of potential dangera pathogen and/or its products--by host cells and molecules. Recruitment and/or generation of destructive effector mechanisms for containment or elimination of the potential danger. Example: Dendritic cells (antigen presenting) Describe the main features of the adaptive immune response, including specificity and memory For INNATE Immune Response: A limited number of binding specificities (distinct receptors) are expressed by cells of the innate defense system Recognition and response are rapid--measured in minutes to hours There is no specific memory generated as a consequence of recognition and response For ADAPTIVE Immune Response: Specific responses expressed by cells of adaptive defense system Recognition and response are slow in 4-14 days. Time after InfectionType of ImmunityDefenses Used

ImmediateInnatePhysical and chemical barriers

0 -4 hoursInnateMolecular and cellular recognition and response

4 96 hoursInnateMolecular and cellular recruitment of cells from circulation and their activation

4 14 daysAdaptiveLymphocytes: Specific responses (memory cells are generated)

Determine how the genome can encode for the diverse antigen receptor repertoire utilized by B and T cells to detect a wide array of pathogens Stem cells in bone marrow are the source of T- and B- cells. T cells undergo further maturation in the thymus and are responsible for cell-mediated immunity and regulation of immune responses. B cells, when activated, are responsible for the production of antibodies. In B cells, clonal expansion method is used. Proliferation and diversification in bone marrow first occurs. Then, antigen binds to specific B cell in peripheral lymphoid organ. Last, proliferation (clonal expansion) and differentiation of B cells occurs. Benefit of clonal expansion is that you dont create cells that you dont need When antigen is present, only B cell that is specific for antigen will be activated. Once activated, it will make lots of copies of itself.

Memory also plays a role to detect wide array of pathogens. Memory cells hang out for some time. Nave cell forms effector cells (they go out and neutralize or destroy pathogen) (also form memory cells). Memory cells can now generate more memory cells and effector cells upon another encounter with antigen. This is only seen in innate response.

Alternative splicing gets a lot of products from same gene. Non homologous end-joining In each cell specific to certain pathogen, the DNA will be different among cells

Describe two mechanisms that support immunological tolerance to self antigens Central- Maturing B cells in the bone marrow and T cells in the thymus are screened for expression of potentially auto-reactive receptors. Auto-reactive cells are deletedclonal deletion. Peripheral- In the periphery, auto-reactive B and T cells are inactivated (anergized) clonal inactivation, or are prevented from being activated by inhibitory regulatory T lymphocytes (Treg).Antigen Presentation Explain how T cells see antigen, and how this is different from how antibodies detect antigen T cells only see the processed peptide loaded on the MHC The structure of TCRs is analogous to immunoglobulins, but it is always cell-bound and never secreted (like antibodies from B cells are) TCRs must recognize antigen presented by specialized cells; antibodies recognize antigens in the milieu (or environment) TCRs recognize a complex of an antigen-derived peptide epitope AND portions of the presenting, self MHC molecule T cells see antigenic determinants via their antigen specific T cell receptor (TCR) TCRs recognize linear antigenic peptide epitopes These epitopes do not exist in the native antigen structure T cell receptors recognize antigenic peptide epitopes presented by MHC molecules processed polypeptide epitopes are brought to and expressed at the surface of antigen presenting cells by MHC class I or MHC class II presenting molecules T cell sees chewed up peptide of bacteria. Doesnt see whole bacteria. Antibodies see epitope on surface of bacteria in its native form. Define "major histocompatibility complex (MHC)" and its role in the immune response. They were originally defined as a major barrier to organ and tissue transplantation, since they are all encoded by extremely polymorphic genetic loci in humansthe major histocompatibility complex Describe the difference between Class I and Class II MHC molecules - what cells express them and the role they play in defense. MHC class I molecules expressed on antigen presenting cells are required for presentation of antigen to and activation of CD8+ cytotoxic T cells MHC class I molecules are expressed on all cells of the body except red blood cells. MHC class II expressed on antigen presenting cells are required for presentation of antigen to and activation of CD4+ helper T cells MHC class II molecules are only expressed by antigen presenting cells (dendritic cells, monocytes/tissue macrophage and B cells) Both classes of MHC molecules bind and present antigenic peptides to T cell antigen receptors Suggest why CD8 T cells tend to respond more to intracellular antigens whereas CD4 T cells tend to respond to extracellular antigens

Autoimmunity Case Study Discussion: Celiac Disease Define, draw, and explain what an antibody is An antibody is a large protein that consists of four interlinked peptides. The antigen binding sites work in a ligand-receptor or lock-and-key fashion with antigens that accompany microbial invasion. Two identical light chains 2 or 2. Two identical heavy chains 2m, 2d, 2g1, 2g2, 2g3, 2g4, 2a1, 2a2, or 2e. The heavy chain type defines the class Two 1 chains would define an IgG antibody, and in this case, the subclass IgG1 Two m heavy chains would define an IgM antibody, etc.

Define autoimmune disease and the role of autoantibodies Describe the symptoms, diagnosis, risk factors, and treatments for celiac disease Discuss possible hypotheses for the development of autoimmune disease Explain, at a cellular and molecular level, how celiac disease is thought to be triggered and maintained, including the role of gluten, TG2, B cells, and T cells, and Dendritic cells Suggest why celiac disease challenges previous hypotheses for the development of autoimmunity